The objective of the proposed research is to determine the mechanism by which small molecules affect the stability of hemoglobin. The approach in all phases of this study will be to work first with well defined chemical systems, then with cells and animals. Studies in this laboratory have shown that substituent groups on the benzene ring of phenylhydrazine modify the severity of arylhydrazine-induced anemia and also modify the ability of an arylhydrazine to cause the formation of a specific ferrihemochrome with an exogenous aryl ligand. Arylhydrazines with ring substituents that differ in number, position, size, charge, and electronegativity will be synthesized in order to obtain further knowledge of how these factors affect the induction of anemia and ferrihemochrome. The sequence of events that lead from ferriheomochrone to Heinz body will be followed by chemical studies of prophyrin and globin. The next phase of the proposed research will deal in similar manner with the mechanism of drug-induced hemolytic anemia. The structure of most hemolytic drugs include an aromatic amine component. Metabolites of these amines will be synthesized and tested in hemoglobin solutions, erythrocytes, and animals. Experiments will be conducted to determine the minimum amount of certain compounds that will cause the depletion of glutathione in erythrocytes without the production of Heinz bodies, as well as the minimal maintenance dose of those compounds required to deplete the glutathione of circulating erythrocytes in rabbits. The glutathione-depleted cells and animals will then be tested with known hemolytic drugs. Analogues of these drugs with substituent groups at different positions on the aromatic ring will be synthesized and tested in erythrocytes and animals to determine whether or not the induction of Heinz bodies and anemia can be blocked by structural modifications. In the final phase of the proposed research, the binding of chemically inert ring compounds to hemoglobin by non-polar interactions and the effect of such binding on the stability of hemoglobin will be investigated.